Following basal stem rot in young oil palm plantings

The PCR primer GanET has previously been shown to be suitable for the specific amplification of DNA from Ganoderma boninense. A DNA extraction and PCR method has been developed that allows for the amplification of the G. boninense DNA from environmental samples of oil palm tissue. The GanET primer reaction was used in conjunction with a palm-sampling programme to investigate the possible infection of young palms through cut frond base surfaces. Ganoderma DNA was detected in frond base material at a greater frequency than would be expected by comparison with current infection levels. Comparisons are made between the height of the frond base infected, the number of frond bases infected, and subsequent development of basal stem rot. The preliminary results suggest that the development of basal stem rot may be more likely to occur when young lower frond bases are infected.     

Life expectancy of oil palm (Elaeis guineensis) infected by Ganoderma boninense in coastal soils,Malaysia: a case study

Ganoderma boninense basal stem rot poses a serious threat to the oil palm industry. The effects of external disease symptoms and coastal soils (Briah – Typic Endoaquepts, Jawa – Typic Sulfaquepts, and Selangor – Typic Humaquepts) on the life expectancy of the infected palms, from disease detection to death, were studied. Six-monthly censuses on disease classes for each palm were recorded between 2004 and 2012. Survival curves of disease symptoms and soil types were compared using Kaplan–Meier and log-rank methods, respectively. Ganoderma-infected palms in acid-sulphate (AS) and potential AS soils recorded lower life expectancy. Survival duration of infected palms with foliar symptoms was 12-months shorter. External factors, such as soil type may influence the survival of infected palms and soil types may pre-dispose oil palm to higher risk of Ganoderma infection. More effective Ganoderma management for palms planted on Coastal soils (with and without AS layer) have been proposed.     

In vitro Antagonistic Interactions Between Endophytic Basidiomycetes of Oil Palm (Elaeis guineensis) and Ganoderma boninense

Ganoderma boninense is a white rot basidiomycete that causes basal stem rot disease of oil palm (Elaeis guineensis). The aims of this study were to identify endophytic basidiomycetes occurring naturally within oil palm and to assess their potential as biocontrol agents against G. boninense strain PER71 in vitro. In total, 376 isolates were recovered from samples collected from the root, stem and leaves of oil palm using Ganoderma‐selective medium. Ten of these isolates (2.7% of the total 376 isolates) were identified as basidiomycetes on the basis of clamp connections and the production of poroid basidiomes after incubation in glass jars containing PDA medium for 7–12 days. The isolates were identified using ITS rDNA sequencing as Neonothopanus nambi (five isolates), Schizophyllum commune (four isolates) and Ganoderma orbiforme (one isolate). The N. nambi isolates showed the greatest antagonistic activity against G. boninense, based on 73–85% inhibition of the radial growth measurements of G. boninense in dual culture and 76–100% inhibition of G. boninense growth in a culture filtrate assay. Possible modes of action for the antagonism shown by N. nambi against G. boninense in vitro include competition for substrate availability, space and the production of non‐volatile metabolites or antibiotics that inhibited the growth of G. boninense. Further in vivo investigations are required to determine the ability of N. nambi isolates to colonize oil palm seedlings and to protect oil palm from infection when challenged with G. boninense.     

Molecular biology of <Emphasis Type="Italic">Ganoderma</Emphasis> pathogenicity and diagnosis in coconut seedlings

The pathogenicity of Ganoderma boninense was tested on coconut seedlings under greenhouse conditions and infection confirmed by using immunological and molecular diagnostic tools. Desiccation of older leaves and the emergence of sporophores were observed from pathogen-inoculated seedlings, whereas a control seedling does not show any pathogenic symptoms. Mature sporophores were formed within 10–13 weeks after inoculation. Polyclonal antibodies raised against mycelial proteins of Ganoderma were used for detection of Ganoderma in infected field palm and seedlings through indirect enzyme-linked immunosorbent assay technique. We adopted dot-immunobinding assay for the detection of Ganoderma from greenhouse and field samples. Under nucleic-acid-based diagnosis, G. boninense (167 bp) was detected from artificially inoculated seedlings and infected field palms by polymerase chain reaction. Apart from these, histopathological studies also support the Ganoderma pathogenicity in coconut seedlings. The pathogenicity test and combination of all the three diagnostic methods for Ganoderma could be highly reliable, rapid, sensitive and effective screening of resistance in planting material in the future.     

Stem rots of oil palm caused by <Emphasis Type="Italic">Ganoderma boninense</Emphasis>: Pathogen biology and epidemiology

Oil palm (Elaeis guineensis Jacq.) has been grown in Papua New Guinea since the early 1960s. The most important disease of oil palm in PNG is a stem rot of the palm base. This is the same disease that constitutes a major threat to sustainable oil palm production in SE Asia. Investigations into the causal pathogen have revealed that the stem rots in PNG are caused predominantly by the basidiomycete Ganoderma boninense, with a minor pathogen identified as G. tornatum G. tornatum was found to have a broad host range whereas G. boninense appears to be restricted to palms. The population structure of G. boninense was investigated using inter-fertility studies between isolates collected from basal stem rots on oil palm. Although the G. boninense field populations are predominantly comprised of distinct individuals, a number of isolates were found that share single mating alleles. This indicates that out-crossing had occurred over several generations in the resident or wild population of G. boninense prior to colonization of oil palm. No direct hereditary relationship between isolates on neighbouring diseased palms was found, although an indirect link between isolates causing upper stem rot and basal stem rot was detected.     

Comparing interfertility data with random amplified microsatellites DNA (RAMS) studies in <Emphasis Type="Italic">Ganoderma</Emphasis> Karst. Taxonomy

The taxonomy of the causal pathogen of basal stem rot of oil palms, Ganoderma is somewhat problematic at present. In order to determine the genetic distance relationship between G. boninense isolates and non-boninense isolates, a random amplified microsatellites DNA (RAMS) technique was carried out. The result was then compared with interfertility data of G. boninense that had been determined in previous mating studies to confirm the species of G. boninense. Dendrogram from cluster analysis based on UPGMA of RAMS data showed that two major clusters, I and II which separated at a genetic distance of 0.7935 were generated. Cluster I consisted of all the biological species G. boninense isolates namely CNLB, GSDK 3, PER 71, WD 814, GBL 3, GBL 6, OC, GH 02, 170 SL and 348781 while all non-boninense isolates namely G. ASAM, WRR, TFRI 129, G. RES, GJ, and CNLM were grouped together in cluster II. Although the RAMS markers showed polymorphisms in all the isolates tested, the results obtained were in agreement with the interfertility data. Therefore, the RAMS data could support the interfertility data for the identification of Ganoderma isolates.     

Identification of <Emphasis Type="Italic">Ganoderma</Emphasis>, the causal agent of basal stem rot disease in oil palm using a molecular method

From comparison of the alignments of the internally transcribed spacers (ITS) of ribosomal DNA from Ganoderma associated with oil palm basal stem rot (BSR) and other Ganoderma species, two specific primer pairs were selected to provide a specific DNA amplification of pathogenic Ganoderma in oil palm. Each primer pair produced a single PCR product of about 450 bp (for primer pair IT1–IT2) and 334 bp (for primer pair IT1–IT3) when oil palm Ganoderma DNA was used. No PCR amplification product was observed when other Ganoderma species DNA was used in PCR amplification with these primer pairs. Three specific restriction enzyme sites were identified in the ITS and intergenic spacer (IGS1) regions. The restriction enzymes MluI, SacI and HinfI were used to digest the ITS-PCR product and restriction enzymes TfiI, ScaI and HincII were used to digest the IGS1-PCR product. Of the three restriction enzymes used in each rDNA region, MluI specifically digested the ITS regions, and TfiI specifically digested the IGS1 region of oil palm Ganoderma. Analysis of the published ITS nucleotide sequences of 31 Ganoderma species showed that the MluI restriction site was not present in other Ganoderma species. The use of both specific primers and restriction enzyme analysis can be applied as a standard protocol to identify pathogenic Ganoderma in oil palm. In this study, the use of specific primers and PCR-RFLP analyses of the rDNA gave consistent results for the characterisation of pathogenic Ganoderma, and indicated that Ganoderma strains associated with BSR disease in oil palms belong to a single species.     

Current strategies and perspectives in detection and control of basal stem rot of oil palm

The rapid expansion of oil palm (OP) has led to its emergence as a commodity of strategic global importance. Palm oil is used extensively in food and as a precursor for biodiesel. The oil generates export earnings and bolsters the economy of many countries, particularly Indonesia and Malaysia. However, oil palms are prone to basal stem rot (BSR) caused by Ganoderma boninense which is the most threatening disease of OP. The current control measures for BSR management including cultural practices, mechanical and chemical treatment have not proved satisfactory. Alternative control measures to overcome the G. boninense problem are focused on the use of biological control agents and many potential bioagents were identified with little proven practical application. Planting OP varieties resistant to G. boninense could provide the ideal long-term solution to basal stem rot. The total resistance of palms to G. boninense has not yet been reported, and few examples of partial resistances have been observed. Importantly, basidiospores are now recognized as the method by which the disease is spread, and control methods require to be revaluated because of this phenomenon. Many methods developed to prevent the spread of the disease effectively are only tested at nursery levels and are only reported in national journals inhibiting the development of useful techniques globally. The initial procedures employed by the fungus to infect the OP require consideration in terms of the physiology of the growth of the fungus and its possible control. This review assesses critically the progress that has been made in BSR development and management in OP.     

Development and evaluation of an enzyme-linked immunosorbent assay (ELISA) and dot immunobinding assay (DIBA) for the detection of Ganoderma infecting palms

The genus Ganoderma has a worldwide distribution causing root and stem rot of many plantation crops. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study, we developed polyclonal antiserum for Ganoderma mycelial and extracellular protein, and evaluated its efficacy with different plant samples collected from artificially inoculated coconut seedlings and Ganoderma infected field palms. We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antisera developed against the crude mycelial protein (CMP) and extracellular protein (ECP) showed a 1:1000 titre value for the detection of Ganoderma. The CMP antisera developed showed more cross-reaction when compared to ECP antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In the DIBA test, ECP antisera detected positive control (ECP of Ganoderma MTP and CRS-1 isolate), artificially inoculated roots, infected field roots, infected basal trunk and additionally lesions gave positive reactions which were not found in the CMP antisera tested. Therefore, both ELISA and DIBA tests may be useful for screening a large number of samples and help in the detection of infection at the earliest stage of disease development and this will certainly help to adopt suitable management strategies against Ganoderma disease in palm crops in advance.     

Association of Copper and Zinc Levels in Oil Palm (Elaeis guineensis) to the Spatial Distribution of Ganoderma Species in the Plantations on Peat

Nutrients are essential for normal physiological processes in plants, and they play important roles in defence mechanisms against pathogens. Oil palms cultivated on peat are more prone to nutrient deficiency, especially micronutrients, and this may affect their susceptibility to Ganoderma species, the major threat to the sustainability of oil palm throughout South‐East Asia. This study was conducted to investigate the association of copper (Cu) and zinc (Zn) in mature oil palm to the spatial distribution of Ganoderma species in the plantations on peat. Foliar samples (frond 17) of oil palm from two plantations (Betong and Miri) on peat in Sarawak, Malaysia, were collected based on the spatial distribution pattern of Ganoderma, and total Cu and Zn were quantified spectrometrically. The experiment was conducted twice at a 1‐year interval. The concentrations of Cu and Zn were significantly lower in oil palms from infected areas in contrast to those from uninfected areas. In addition, oil palms in infected areas in Miri suffered Cu and Zn deficiencies. Furthermore, Cu and Zn were significantly lower in the oil palms in Miri that had higher Ganoderma occurrence, as compared to those in Betong, which had significantly higher Cu and Zn but lower Ganoderma occurrence.     

An insight into spore dispersal of <Emphasis Type="Italic">Ganoderma boninense</Emphasis> on oil palm

The disease of oil palm caused by Ganoderma boninense, although universally referred to as Ganoderma basal stem rot, occurs in three very distinct phases, with basal stem rot only part of the disease cycle. G. boninense also causes a seedling disease and an upper stem rot. An understanding of spore dispersal provides an insight into where spores of G. boninense have a role in the infection process. This role will be discussed in relation to each of these three infection phases. This understanding is a critical component of developing a successful disease control strategy.     

Enhancing biological control of basal stem rot disease (<Emphasis Type="Italic">Ganoderma boninense</Emphasis>) in oil palm plantations

Basal Stem Rot (BSR) disease caused by Ganoderma boninense is the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride, Pseudomonas fluorescens, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.     

Characterization of <Emphasis Type="Italic">Streptomyces</Emphasis> spp. isolated from the rhizosphere of oil palm and evaluation of their ability to suppress basal stem rot disease in oil palm seedlings when applied as powder formulations in a glasshouse trial

Ganoderma boninense, the main causal agent of oil palm (Elaeis guineensis) basal stem rot (BSR), severely reduces oil palm yields around the world. To reduce reliance on fungicide applications to control BSR, we are investigating the efficacy of alternative control methods, such as the application of biological control agents. In this study, we used four Streptomyces-like actinomycetes (isolates AGA43, AGA48, AGA347 and AGA506) that had been isolated from the oil palm rhizosphere and screened for antagonism towards G. boninense in a previous study. The aim of this study was to characterize these four isolates and then to assess their ability to suppress BSR in oil palm seedlings when applied individually to the soil in a vermiculite powder formulation. Analysis of partial 16S rRNA gene sequences (512 bp) revealed that the isolates exhibited a very high level of sequence similarity (>?98%) with GenBank reference sequences. Isolates AGA347 and AGA506 showed 99% similarity with Streptomyces hygroscopicus subsp. hygroscopicus and Streptomyces ahygroscopicus, respectively. Isolates AGA43 and AGA48 also belonged to the Streptomyces genus. The most effective formulation, AGA347, reduced BSR in seedlings by 73.1%. Formulations using the known antifungal producer Streptomyces noursei, AGA043, AGA048 or AGA506 reduced BSR by 47.4, 30.1, 54.8 and 44.1%, respectively. This glasshouse trial indicates that these Streptomyces spp. show promise as potential biological control agents against Ganoderma in oil palm. Further investigations are needed to determine the mechanism of antagonism and to increase the shelf life of Streptomyces formulations.     

Early immunodiagnosis of Ganoderma infecting coconut seedlings and palms by using monospecific polyclonal antibodies

Among the various fungal diseases affecting plantation crops viz., coconut, aracanut, oil palm, etc. in India, basal stem rot (BSR) caused by species of Ganoderma is the most destructive. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. In this study we generated two different types of antiserum for diagnosis of Ganoderma using the purified monospecific protein (62 kDa) (MS) and crude sporophore extract (SE). We also tested the cross-reactivity with the soil-borne and saprophytic fungus collected from different parts of coconut palm. The antiserum developed against the MS and SE showed 1:700 and 1:3000 titre values for the detection of Ganoderma. The MS antisera developed showed very low or almost no cross-reaction when compared to SE antisera of Ganoderma. In the DIBA test, at a 1:10 dilution of antigen, 1:1000 dilution of CMP and ECP antisera, 1:5000 dilution of secondary antibody gave clear distinctions in colour development between healthy and diseased samples. In DIBA test, both types of antisera were used separately for pathogenicity tests. MS antisera showed a positive reaction for purified protein, artificially infected roots and infected field palm. A mild reaction was observed against infected field trunk but a negative reaction was observed for lesions and leaf samples. In the case of SE antisera, a negative reaction was observed for all leaf samples, healthy roots and healthy trunk samples but positive reactions were observed for positive control, artificially inoculated roots, infected field roots, infected trunk and lesions samples. Therefore, both ELISA and DIBA tests may be useful in the detection of infection at the earliest stage of disease development and this will certainly help in the development of management strategies against Ganoderma disease in palm crops in advance.     

Ganoderma – a basal stem rot disease of coconut palm in south Asia and Asia pacific regions

Basal stem rot (BSR) caused by the species of Ganoderma is one of the most devastating diseases of numerous perennial, coniferous and palmaceous hosts. In forest systems, Ganoderma has an ecological role in the breakdown or delignification of woody plants. Symptoms of BSR disease can take several years to develop, and the presence of the pathogen (such as indicated by fruiting bodies) is often only visible when the fungus is well established and more than half of the bole tissue has been decayed, leaving no chance for the grower to cure the infected palms. Soils with poor drainage and water stagnation during rainy seasons were found to favour the disease. A limiting factor in controlling the BSR disease is the lack of reliable diagnostic method(s) for early diagnosis. Amendment of calcium nitrate in soil is one of the methods for the management of Ganoderma disease in palms. A combination of biological and systemic fungicides along with good cultural practices will effectively control the BSR disease in palms.     

Automated,high-throughput DNA extraction protocol for disposable label free,microfluidics integrating DNA biosensor for oil palm pathogen,Ganoderma boninense

Basal Stem Rot and Upper Stem Rot diseases caused by pathogenic fungus Ganoderma boninense continue to be a major plight in the palm oil industry. Despite continuous research in combating the problem, resolution remains stagnant. Here, developed an automated, high-throughput DNA extraction protocol on microfluidics device for a quick, disposable, label-free detection, within 2 h of assessment. Microfluidics was designed using AutoCAD software, fabricated on microscopic glass substrate using negative photoresist (SU-8 2015) and molded with a biopolymer silicone, Polydimethylsiloxane. G. boninense and unknown pathogenic fungus isolated from rotten mushroom were grown and fractions of extracted DNA were pooled and analyzed for comparison along with synthetic ssDNA of G. boninense. Results from LPM and HPM show successful fabrication with ≤0.1 mm variance between the dimensions in the design before and after lithography process. The PDMS microfluidics show no leakage when run with DNA samples. Analyses from I-V measurement, UV–vis, FTIR, and PCR show comparable results between extracted and synthetic ssDNA of G. boninense and a contrast with the unknown pathogenic fungus, indicating a successful DNA extraction protocol via microfluidics for label-free identification of G. boninense. Optimization of DNA extraction can be further devised for applicability on lab-on-a-chip devices.     

Morphological and transcript changes in the biosynthesis of lignin in oil palm (Elaeis guineensis) during Ganoderma boninense infections in vitro

Lignification of the plant cell wall could serve as the first line of defense against pathogen attack, but the molecular mechanisms of virulence and disease between oil palm and Ganoderma boninense are poorly understood. This study presents the biochemical, histochemical, enzymology and gene expression evidences of enhanced lignin biosynthesis in young oil palm as a response to G. boninense (GBLS strain). Comparative studies with control (T1), wounded (T2) and infected (T3) oil palm plantlets showed significant accumulation of total lignin content and monolignol derivatives (syringaldehyde and vanillin). These derivatives were deposited on the epidermal cell wall of infected plants. Moreover, substantial differences were detected in the activities of enzyme and relative expressions of genes encoding phenylalanine ammonia lyase (EC 4.3.1.24), cinnamate 4‐hydroxylase (EC 1.14.13.11), caffeic acid O‐methyltransferase (EC 2.1.1.68) and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195). These enzymes are key intermediates dedicated to the biosynthesis of lignin monomers, the guaicyl (G), syringyl (S) and ρ‐hydroxyphenyl (H) subunits. Results confirmed an early, biphasic and transient positive induction of all gene intermediates, except for CAD enzyme activities. These differences were visualized by anatomical and metabolic changes in the profile of lignin in the oil palm plantlets such as low G lignin, indicating a potential mechanism for enhanced susceptibility toward G. boninense infection.